1. MOLECULAR GENETIC RELATIONSHIPS OF THE ZOKORS (RODENTIA, MYOSPALACINAE): ANALYSIS OF D-LOOP REGION POLIMORPHISM Tsvirka Marina 1, Pavlenko Marina 1, Korablev Vladimir 1, Pang Junfeng 2 1 Institute of Biology and Soil Science, Far Eastern Branch Russian Academy of Sciences, Vladivostok, 690022, Russia 2 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, China

3. PROBLEMS Among others subterranean rodents, zokors (Myospalacinae) are still remains poorly studied in viewpoint of taxonomy and evolution. Recent molecular phylogenetic studies have demonstrated that zokors are closely related to Spalacinae and Rhyzomyinae in the family Spalacidae Among others subterranean rodents, zokors (Myospalacinae) are still remains poorly studied in viewpoint of taxonomy and evolution. Recent molecular phylogenetic studies have demonstrated that zokors are closely related to Spalacinae and Rhyzomyinae in the family Spalacidae (Norris et. al, 2004; Jansa, Weksler, 2004) However relationships among the species within Myospalacinae are not clearly understood. However relationships among the species within Myospalacinae are not clearly understood.

4. PROBLEMS Genus Myospalax Subgenus Myospalax M. myospalax M. aspalax M. psilurus Subgenus Eospalax M. rothschildi M. smithii M. fontanieri ( M. f.cansus, M. f. rufescens, M. f. baileyi) (Corbet, 1991) Genus Myospalax M. myospalax M. aspalax M. psilurus Genus Eospalax M. rothschildi M. smithii M. fontanieri M. cansus M. rufescens M. baileyi (Zheng, 1994 )

5. Map of Zokors Distribution in the Russia M. myospalax M. aspalax M. psilurus M. armandii 2n=44 2n=62 2n=64

6. Background Professor N. N., Vorontsov prominent Russian biologist was initiator of study of fossorial rodents in Russia and adjacent countries Professor N. N., Vorontsov prominent Russian biologist was initiator of study of fossorial rodents in Russia and adjacent countries L. Martynova, N. Vorontsov, 1975. Population cytogenetic of Zokors Chromosomes L. Martynova, N. Vorontsov, 1975. Population cytogenetic of Zokors Chromosomes L. Martynova, 1976. Chromosomal differentiation of three species of zokors. L. Martynova, 1976. Chromosomal differentiation of three species of zokors. L. Martynova, I. Fomicheva, N. Vorontsov, 1977. Electrophoretic study of blood protein of zokors. L. Martynova, I. Fomicheva, N. Vorontsov, 1977. Electrophoretic study of blood protein of zokors.

7. Electrophoretic patterns of transferrin (TF) of zokors (Pavlenko & Korablev, 2003; 2005) 1. Tf-A – M. aspalax, Transbaikalia 2. Tf-B – M. psilurus, Primorye 3. Tf-C – M. psilurus, Transbaikalia 4. Tf-C1 – M. armandii 5. Tf-D – M. myospalax, Altai region 12345

8. M. smithii M. rufescens 2n=62 M. rothschildi M. fontanieri M. baileyi M. cansus Map of Zokors Distribution in the China 2n=62

9. C. Zhou and K. Zhou. The validity of different zokor species and the genus Eospalax inferred from mitochondrial gene sequences / Integrative Zoology 2008; 3: 290–298. C. Zhou and K. Zhou. The validity of different zokor species and the genus Eospalax inferred from mitochondrial gene sequences / Integrative Zoology 2008; 3: 290–298. Myospalax and Eospalax genera

10. Phylogenetic relationships of zokors based on the RAPD- PCR analysis(Tsvirka et al., 2009) Phylogenetic relationships of zokors based on the RAPD- PCR analysis (Tsvirka et al., 2009) M. psilurus M. smithii M. aspalax M. armandii M. myospalax 4 species groups within genus Myospalax 100 74 100 54

11. Puzachenko A., Pavlenko M., Korablev V. Craniological variability of the zokors (Myospalacinae) with simplified first upper molar. Abstracts of 11 International Conference "Rodens et Spatium", 24-28 July 2008, Myshkin (Russia). P.144 Puzachenko A., Pavlenko M., Korablev V. // Zool. Z. 2009. V. 88. №1. P. 92-112. Puzachenko A., Pavlenko M., Korablev V. Craniological variability of the zokors (Myospalacinae) with simplified first upper molar. Abstracts of 11 International Conference "Rodens et Spatium", 24-28 July 2008, Myshkin (Russia). P.144 Puzachenko A., Pavlenko M., Korablev V. Variability of skulls in Zokors (Rodentia,Miospalacinae) // Zool. Z. 2009. V. 88. №1. P. 92-112. 3 species groups with taxonomic ranks of the genera or subgenera: M. myospalax; M. aspalax and M. armandii; M. smithii and M. psilurus

12. The aims of the present study were to determine the systematic position of Myospalax and Eospalax;to determine the systematic position of Myospalax and Eospalax; to clarify the phylogenetic relationships among zokor species;to clarify the phylogenetic relationships among zokor species; to confirm or contradict the taxonomic position of M. armandii as a distinct species;to confirm or contradict the taxonomic position of M. armandii as a distinct species; to confirm or disprove previous data concerning genetic differentiation of Manchurian zokors M. psilurus from Khanka Plain and Transbaikalia.to confirm or disprove previous data concerning genetic differentiation of Manchurian zokors M. psilurus from Khanka Plain and Transbaikalia.

13. 54 specimens of 7 species from 30 different localities in Russia and China. 1 specimen of Spalax judaei from GenBank was used as outgroup.MATERIAL M. cansus

14. METHODS Phylogenetic reconstructions using MEGA 4 (Tamura et al., 2007): Neighbor-Joining (NJ), Maximum Parsimony (MP), Minimum Evolution (ME), unweighted pair-group method with arithmetic averaging (UPGMA) ( Calculation of genetic distances based on Kimura two-parametric (K2P) estimator. Sequencing 5’ end of the D-loop

15. RESULTS The alignment of the mt DNA sequences of 7 taxa comprises 518 nucleotides.The alignment of the mt DNA sequences of 7 taxa comprises 518 nucleotides. 206 (40%) nucleotides were variable.206 (40%) nucleotides were variable. 187 (36%) nucleotides were parsimony informative.187 (36%) nucleotides were parsimony informative. The average ratio of transition/transvertion was 3.58.The average ratio of transition/transvertion was 3.58.

16. NJ and UPGMA trees of Zokors based on the D-loop sequences (MEGA-4.1) M. myospalax M. smithii M. cansus M. rufescens M. aspalax M. armandii M. psilurus Primorye M. psilurus Zabaykalye M. psilurus

17. Interspecific relationships within the genus Myospalax 12345678 1. M. cansus - 2. M. smithii 0.120- 3. M. rufescens 0.123 0.062- 4. M. aspalax 0.190 0.167 0.164 - 5. M. armandii 0.185 0.181 0.070 - 6. M. myospalax 0.225 0.226 0. 224 0.1770.190- 7. M. psilurus (П) 0.158 0.154 0.154 0.076 0.084 0.159 - 8. M. psilurus (З) 0.164 0.160 0.108 0.104 0.181 0.063- Estimate value of Kimura two-parametric distance matrix for 8 taxa of Myospalax

18. DistancesRAPDD-loop within species0.050.003-0.028 within forms of M. psilurus0.04-0.050.003 between species (M. aspalax - M. armandii M. smithii - M. rufescens) 0.080.062- 0.07 between two forms of M. psilurus 0.080.063 between distant species (Myospalax – Eospalax) 0.25-0.350.154-0.226 between genera (Myospalax – Spalax) 0.310.238-0.304 Genetic distances for nuclear (RAPD) and mitochondrial (D-loop) DNA

19. Conclusions Our results didn't confirm the hypothesis of validity of the genus Eospalax suggested by Zhou and Zhou (2009). The all studied zokor species should be divided into 3 species groups within one genus Myospalax: "myospalax", "psilurus-armandii-aspalax" and "smithii-cansus-rufescens". Our results didn't confirm the hypothesis of validity of the genus Eospalax suggested by Zhou and Zhou (2009). The all studied zokor species should be divided into 3 species groups within one genus Myospalax: "myospalax", "psilurus-armandii-aspalax" and "smithii-cansus-rufescens". The species within the "psilurus-armandii-aspalax" (M. psilurus, M. armandii, M. aspalax) as well as within "smithii-cansus-rufescens" (M. smithii, M. cansus, M. rufescens) groups were closely related among themselves. The M. myospalax, differs from the other species in chromosomal and molecular features, is more closely related with the first species group than the second one. The species within the "psilurus-armandii-aspalax" (M. psilurus, M. armandii, M. aspalax) as well as within "smithii-cansus-rufescens" (M. smithii, M. cansus, M. rufescens) groups were closely related among themselves. The M. myospalax, differs from the other species in chromosomal and molecular features, is more closely related with the first species group than the second one. The hypothesis of species status of M. aspalax suggested on craniometrical analysis of zokors were confirmed (Puzachenko et al., 2009). The hypothesis of species status of M. aspalax suggested on craniometrical analysis of zokors were confirmed (Puzachenko et al., 2009). The hypothesis about the independent species status of two geographical populations of M. psilurus from Khanka Plain and Transbaikalia earlier suggested on t biochemical and karyological data (Pavlenko, Korablev, 2003) was also confirmed on the results of mt DNA analysis. The hypothesis about the independent species status of two geographical populations of M. psilurus from Khanka Plain and Transbaikalia earlier suggested on t biochemical and karyological data (Pavlenko, Korablev, 2003) was also confirmed on the results of mt DNA analysis.

20. The Authors Marina Pavlenko Vladimir KorablevJunfeng Pang Marina Tsvirka

21. (grant nos. (grant nos. The study was supported by the RFBR (grant nos. 06-04- 39015) and FEBRAS (grant nos. s 09-III-В-138). ACKNOWLEDGMENTS